The overall goal of this procedure is to provide an automated method to analyze and quantify changes in morphology of an undefined shape cell taken from three dimensional confocal fluorescence images. This is accomplished by first performing the chemical stimulation experiment to include cells treated with agonist cells, treated with antagonist, followed by agonist and cells with no treatment. The cells are then prepared for immunofluorescence analysis.
The second step is to acquire multi-spectral three-dimensional fluorescence images. Next three-dimensional morphometric analysis is performed using ameris neuroscience, ameris XT, and MATLAB computer software. The single cell phenotypic changes visualized through three dimensional morphometric analysis are then analyzed and quantified as a final step.
Ultimately, this software platform is used to quantify changes in cell shape following receptor activation, providing an additional tool for drug discovery. Generally, researcher have a standard expertise in biological system, may not have familiarity to computer application. In this protocol in I 60 module, bridge the gap between de visualization and computer language Prior to the start of this procedure.
Transfect human embryonic kidney cells with hemagglutinin tagged corticotropin releasing factor receptor two or CRF R two as referenced in the written protocol. CFR two is a G protein coupled receptor or GPCR. The day after the transfection flame cover slips and place them into a six well plate.
Add 10 milliliters of PBS to a vial of five milligrams of lyophilized polylysine and mix. Then dilute five milliliters of dissolved polylysine in 500 milliliters of PBS and add two milliliters of the mixture onto each of the cover slips. Leave the plate with cover slips at room temperature for 20 minutes After 20 minutes, wash the cover slips by adding two milliliters of PBS and then removing, repeat this process twice.
Following edition of two milliliters of DMEM with 10%FBS media add two to five drops of cells onto the cover slips. The cells should be at the necessary concentration to achieve 60%co fluency. The next day.
Incubate the cells overnight at 37 degrees Celsius the following day. Check that the cells are 60%confluent for agonist treatment. Stimulate designated cells by replacing media with two milliliters of media supplemented with the CRF R two endogenous ligand corticotropin releasing factor at a concentration of one micromolar.
Incubate the cells in a 37 degrees Celsius incubator for 30 minutes for cells to be pretreated with antagonist. Replace media with two milliliters of media supplemented with the selective CFR two antagonist anti salvaging 30 at a concentration of one micromolar. Incubate the cells in a 37 degree Celsius incubator for 30 minutes.
After antagonist treatment. Stimulate the cells with the agonist CRF and incubate it 37 degrees Celsius for 30 minutes for untreated cells. Replace media with two milliliters of fresh media following treatment.
Replace the media in each well with two milliliters of fresh media and add anti HHA diluted one to a thousand, subsequent to a 60 minute incubation at 37 degrees Celsius. Aspirate the media and add two milliliters of fixative to each. Well incubate the plate at room temperature for 20 minutes after aspirating the fixative.
Wash the cells three times with tris puffered saline.Cain. Add 100 microliters of blood solution on top of each cover, slip and incubated room temperature for 30 minutes. Then aspirate the existing blotto solution from the wells and add 100 microliters of fresh secondary antibody cocktail onto each cover slip following a 45 minute incubation at room temperature in the dark wash four times with TBSC by gently adding and removing the solution from the sidewall of the well while still in the final wash solution.
Pick up each cover slip with a needle and curved forceps. Place the cover slip cells facing down onto a slide with a drop of vector shield mounting medium with dappy fix with nail polish and let it dry for 15 to 20 minutes. Alexa, 4 88 nanometer conjugate muse antibody is used to visualize CFR two and DAPI is used to visualize the nuclei mitotic stage to begin mount the fix cells onto a fluorescent microscope to limit the experimental subjectivity, keep the experimental conditions unknown until after the images are acquired and analyzed to acquire images.
A plan acro mat oil, DIC objective with 63 x magnification and 1.4 numerical aperture was used in combination with the zeis LSM five 10 meta confocal microscope connected to a coherent integrated two photon laser system During the data acquisition process, compartmentalize the cells both by multichannel and c partitioning to include data from the nuclear membrane to the outer extracellular receptor extremities. Process the fluorescence data using amris, which allows the visualization and segmentation of a 3D microscopy dataset. Following the algorithm designed by Amaris first, utilize the surface rendering to represent the NU nuclear membrane.
Amaris will determine if there is more than one nucleus in the region of interest, or ROI. Next utilize the spots creation algorithm to locate the CFR two extension spots. Compensates for background noise and irregular intensity of the complex network of amorphous shaped cells to maximize the inclusion of each unit of fluorescence detection of CFR two, set the spots diameter to 0.2 micrometers, which is the smallest unit within the image.
This allows for the extrapolation of distinct information in the form of a measured intensity. Using a Gaussian filter, incorporate spot filtering in the spot's automated creation process. To avoid data cation, convert the dataset from eight bit fixed point to 32 bit float.
Select the created surface and determine the exact spatial location of each spot outside the nuclear surface by performing a distance transformation using the nuclear membrane as a reference point. Exchange the voxel intensities data to spot coordinates data using ameris XT module interfaced with MATLAB. Select spots and select statistical coded in statistical type.
Choose the intensity center reported in the new channel created. Load the desired color for display and set the color map range for analysis. The numerical data can be visualized in the statistical tab and exported to excel using GraphPad prism.
Quantify the resulting data and present in graphical format for statistical analysis. Perform comparisons between groups using two-way Inova and Bonferroni post-test present data as mean plus or minus standard deviation. To demonstrate the power of this approach, the cellular changes that result from the interaction of G-protein coupled receptors and corticotropin releasing factor receptor two with its endogenous ligand CRF.
In transfected HT K 2 93 cells were quantified merged images are visualized using Alexa 4 88 conjugated, anti mouse, secondary antibody and DPI to visualize the nuclei. The results reveal that CRF R two receptors are located in the plasma membrane and project from finite regions of the membrane of the cells. Using conventional 2D analysis, it is possible to detect this subset of extracellular CRF R two receptors only if the receptor adhesion points on the glass covers are analyzed.
Consequently, any other information derived from ZS stacked MULTISPECTRAL data is lost. The result chose no difference between no treatment and agonist. While the receptor adhesion points are dramatically different when the cells are treated with CRF, the extracellular receptors are greatly reduced as shown by the decrease in distance of the spots from the plasma membrane.
They're also redistributed from primarily finite locations into a number of discreet locations. The effect of CRF on receptor membrane distribution is prevented by pretreatment with the CR two specific antagonists. 30 30.
Under these conditions, the CRFR two extensions do not change with CRF treatment. The distal distribution of spots plotted in five micrometer spectrum color-coded intervals is utilized to visualize the distance of the voxels from the nuclear membrane. No treatment and antagonist pre-treatment before agonist treatment shown no significant difference in GPCR contraction.
Treatment of the cells with the agonist CRF progressively reduces the number of CFR two containing voxels when compared to no treatment or as compared to cells. Pretreated with antagonist As after his development. This technique played the way for researcher in the field of quantification imaging technique to explore and characterize numerous single cell phenotypic changes making this cell-based assay an additional single cell profiling tool for the drug discovery process.
